The proper interplay between the nuclear lamina and NPCs is essential for coordinating overall nuclear organization, structure, and function [
9,
16]. Recent studies provide multiple lines of evidence that NPC and NCT abnormalities can result from the C9orf72 HRE [
3,
7,
10,
14,
36]. However, to date, overall nuclear morphology has yet to be thoroughly examined in disease relevant cell types. Despite prior work examining the nuclear lamina in G
4C
2 and DPR overexpression based model systems [
18,
37], which concluded that there were nuclear membrane alterations in those models, our current analyses using a large number of iPSC lines and postmortem patient tissues suggest that there are in fact no overt alterations to overall nuclear morphology or the frequency of Lamin B1 invaginations in C9orf72 ALS/FTD. While a previous study in postmortem tissue characterized nuclear morphology on the basis of “abnormal” vs “normal” [
29], here we comprehensively evaluated nuclear shape in 2D and 3D using measures of circularity and sphericity in iPSNs differentiated from young and aged iPSCs as well as postmortem motor cortex. Employing our more standardized and robust analysis, the overall conclusion that nuclear morphology is unaltered in C9orf72 ALS/FTD (Figs. 1, 2, 3, 4) is consistent with prior studies [
29].
In addition to our global morphology analyses, we examined nuclear lamina pathology in iPSNs and postmortem motor cortex. Based on prior work from diseases caused by mutations in nuclear lamins (laminopathies), nuclear lamina pathology is observed as an invagination of the nuclear envelope [
15,
35]. In our current study, invaginations were defined as area where Lamin B1 immunoreactivity extended partway into the nucleoplasm before retreating back towards the “nuclear rim.” Invaginations differ from nuclear folds in that a fold can be observed as an area of lamin staining which extends from one side of the nuclear rim to the other side thereby forming a complete “bridge” across the entirety of the nucleus. In fact, it has been previously described that multiple lamin staining patterns including “rim”, “punctate”, “diffuse”, and “folded” can be observed in both control and C9orf72 ALS/FTD postmortem tissues [
29]. Consistent with that prior 2D study in a small number of postmortem patient tissues [
29], the nuclear lamina of human neurons appears to form a complex topology with many apparent “folds” along the 3D nuclear surface (Figs.
1a,
2a). While the biological function of this folded lamina patterning is not yet understood, the possibility remains that some previous reports of nuclear lamina disruptions in iPSNs [
24] simply reflect the normal folding along the 3D surface and edges of neuronal nuclei. Therefore, it is important to carefully consider whether nuclear lamina abnormalities reported are truly pathological invaginations such as those known to occur in laminopathies [
15,
35]. To avoid errors in classification, it is essential to evaluate the entire nucleus in 3D space in order to properly distinguish between an invagination and normal nuclear curvature. Prior studies in both non-neuronal cell lines [
1,
7,
18,
37] and postmortem tissues [
29] appear to misclassify “normal” nuclear curvatures as “folding” pathology. Despite this misclassification, multiple groups have evaluated 2D images and reported an increase in nuclear folding in C9orf72 ALS/FTD model systems [
1,
7,
18,
37]. However, all of these studies were conducted using massive overexpression of the C9orf72 HRE or individual DPRs which often accumulate in the perinuclear space [
29,
34]. As nuclear membrane morphology such as overall curvature, invaginations, or folding can be impacted by increased mechanical force from actin fibers [
12,
15], it is plausible that massive artificial overexpression triggers an increase in mechanical pressure on the nucleus. In contrast, our current analysis thoroughly examines true nuclear lamina invagination pathology by accurately tracing Lamin B1 fibers in 2D and 3D space in endogenous model systems.
Consistent with previous reports that aged iPSCs display nuclear lamina alterations reminiscent of those observed in laminopathies and cellular senescence [
25], we found that Lamin B1 invaginations increased with iPSC passage number in both control and C9orf72 iPSNs. Interestingly, NCT capacity and the expression of nuclear transport proteins decline with age [
4,
22,
23,
26] and inhibition of nuclear import can impact nuclear lamins [
13]. Therefore, a plausible hypothesis is that increased frequency of Lamin B1 invaginations is related to normal age related decline in NCT. Nonetheless, these studies, as well as our own, highlight the critical importance of matching passage number between cell lines when examining nuclear biology in iPSC based neurodegenerative disease models.